Biologically active peptides and compositions, their use

ABSTRACT

Derivatives of hemiasterlin or Geodiamolide G having anti-mitotic activities and useful in treating cancer. These derivatives are represented by formula I 
                         
wherein Y, n, R 1 , R 2 , R 3 , R 7 , R 70 , R 71 , R 72 , R 74 , and R 75  are as defined in the specification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 09/593,417,filed Jun. 14, 2000, now U.S. Pat. No. 6,870,028 which is a divisionalof U.S. patent application Ser. No. 08/930,584, filed Feb. 2, 1998, nowU.S. Pat. No. 6,153,590, issued Nov. 28, 2000, which in turn is the U.S.National Stage of PCT/GB96/00942, filed Apr. 22, 1996. The disclosuresof all prior applications are hereby incorporated herein in theirentirety by reference.

This invention relates to novel biologically active compounds andcompositions, their use and derivation.

The invention has involved the extraction of novel biologically activecompounds from marine sponges.

Except where otherwise stated, throughout this specification, any alkylmoiety suitably has up to 8, especially up to 6, most preferably up to4, carbon atoms and may be of straight chain or, where possible, ofbranched chain structure. Generally, methyl is a preferred alkyl group.Halogen atoms may be fluorine, chlorine, bromine or iodine. A preferredacyl group is alkylcarbonyl, especially acetyl.

Except where otherwise stated in this specification, optionalsubstituents of an alkyl group may include halogen atoms, for examplefluorine, chlorine, bromine and iodine atoms, and nitro, cyano, alkoxy,hydroxy, amino, alkylamino, sulphinyl, alkylsulphinyl, sulphonyl,alkylsulphonyl, amido, alkylamido, alkoxycarbonyl, haloalkoxycarbonyland haloalkyl groups. Preferably, optionally substituted alkyl groupsare unsubstituted.

Except where otherwise stated, throughout this specification therecitation of a compound denotes all possible isomers possible withinthe structural formula given for those compounds, in particulargeometrical and optical isomers. Unless otherwise stated definitions areto be regarded as covering mixtures of isomers, and individual isomers,including racemic mixtures, where they can be resolved.

Except if otherwise stated, definitions of compounds in thisspecification are to be regarded as covering all possible salts of thecompounds.

Compounds according to the first aspect of the invention have been foundto be surprisingly effective in the in vivo treatment of cancer.

In accordance with the first aspect of the present invention there isprovided the use of a hemiasterlin compound of general formula

wherein:

-   R₁ and R₇₀ independently represent a hydrogen atom or an optionally    substituted alkyl or acyl group;-   R₂ represents a hydrogen atom or an optionally substituted alkyl or    acyl group or is absent when R₆ represents a group —CH═ as    hereinafter described;-   R₇₃ represents a hydrogen atom or an optional substituent or is    absent when R₆ represents a methylene group or a group —CH═ as    hereinafter described;-   Y represents an optional substituent;-   n represents 0, 1, 2, 3, or 4;-   R₃ represents a hydrogen atom, or an optionally substituted alkyl    group;-   R⁷⁴ represents a hydrogen atom, a hydroxy group or an optionally    substituted alkyl or acyl group;-   R₇ represents a hydrogen atom or an alkyl group;-   R₇₅ represents an optionally substituted alkyl group; and-   i) R₆ and R₇₁ independently represent a hydrogen atom or an    optionally substituted alkyl or acyl group; and-   R₇₂ represents a hydrogen atom;-   or-   ii) R₇₁ represents a hydrogen atom or an optionally substituted    alkyl or acyl group and R₇₂ represents a hydrogen atom or R₇₁ and    R₇₂ each represent radicals so that a double bond is formed between    the carbon atoms to which they are attached; and-   R₆ represents an optionally substituted methylene group bonded to    the indole moiety thereby to form a tricyclic moiety; or-   R₆ represents an optionally substituted group —CH═ bonded to the    indole moiety thereby to form an aromatic tricyclic moiety; for the    manufacture of a medicament for use in therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of antimitotic activity of Hemiasterlins withthat of known antimitotic agents.

FIG. 2 shows the antimitotic activity of chemically modifiedHemiasterlins.

DETAILED DESCRIPTION OF THE INVENTION

Preferably, R¹ represents a hydrogen atom or an alkyl group, especiallya methyl group. More preferably, R₁ represents a hydrogen atom.

Suitably, R₂ represents a hydrogen atom or an acyl group. An acyl groupmay be a benzoyl group, but is preferably an alkylcarbonyl group. Anespecially preferred acyl group is an acetyl group. Preferably, R₂represents a hydrogen atom.

Preferably, R₇₀ represents a hydrogen atom or an alkyl group, especiallya methyl group.

Where Y and/or R₇₃ represent optional substituents, said substituentsmay be independently selected from halogen, especially fluorine,chlorine, bromine and iodine atoms and alkyl, acyl, —OH, —O-alkyl,O-acyl, —NH₂, —NH-alkyl, —N(alkyl)₂, —NH-acyl, —NO₂, —SH, —S-alkyl and—S-acyl, wherein the alkyl and acyl groups of the substituents areoptionally substituted.

Preferred optional substituents represented by Y and/or R₇₃ are alkylgroups.

Preferably, R₇₃ represents a hydrogen atom.

Preferably, n represents 0, 1 or 2. More preferably, n represents 0.

Suitably, R₃ represents an alkyl group. Preferably, R₃ represents aC₃₋₆, especially C₃₋₄, branched alkyl group, for example tertiary butylor isopropyl.

Suitably, R₇₄ represents a hydrogen atom or methyl group, especially ahydrogen atom.

Suitably, R₇ represents an alkyl, preferably methyl, group.

Preferably, R₆ represents a hydrogen atom, or an optionally substitutedalkyl group, or a methylene group

bonded to the indole moiety thereby to form a tricyclic moiety. Morepreferably, R₆ represents an alkyl group.

Preferably, R₇₁ independently represents a hydrogen atom or anoptionally substituted alkyl or acyl group. More preferably, R₇₁represents an alkyl, especially a methyl group.

Preferably, R₆ and R₇₁ are as described in i) above.

R₇₅ may represent a group of general formula

wherein Q represents an optionally substituted —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CHCH—, —CH,C.C.— or phenylene moiety; andX represents a group —OR₈, —SR₈, or —N₉R₁₀ wherein R₈, R₉ and R₁₀independently represent a hydrogen atom or an optionally substitutedalkyl group.

Where Q represents one of the aforesaid optionally substituted acyclicmoieties, the moiety may be substituted by one or more alkyl groups. Aphenylene moiety may be substituted by one or more substituents Y asdescribed above.

Where X represents a group —OR₈, suitably R₈ represents a hydrogen atomor a methyl group. Preferably, R₈ represents a hydrogen atom.

Where X represents a group —NR₉R₁₀, suitably R₉ represents a hydrogenatom or an alkyl group, for example a methyl group and R₁₀ represents asubstituted alkyl group.

Where R₁₀ represents a substituted alkyl group, said group preferablyrepresents a group of general formula —CHR₂₁COOH wherein R₂₁ representsan optionally substituted alkyl group. Preferably, R₂₁ represents agroup which includes at least one nitrogen atom. Preferably, R₂₁represents a group of general formula —(CH₂)_(x)NR₂₂R₂₃ wherein x is aninteger, preferably in the range 1-4, and R₂₂ and R₂₃ independentlyrepresent a hydrogen atom or an optionally substituted alkyl, alkenyl orimine group. Preferably, R₂₂ represents a hydrogen atom and R₂₃represents an imine group —C(NH)NH₂.

Preferably, X represents a group —OR₈, wherein R₈ represents a hydrogenatom.

Preferably, R₇₅ represents a group of general formula

wherein R₄ and R₅ independently represent a hydrogen atom or anoptionally substituted alkyl group; R₇₆ and R₇₇ each represent ahydrogen atom or a radical so that a double bond is formed between thecarbon atoms to which they are attached; and X is as described above.

It has been discovered that compounds of general formula I, can causemitotic arrest and the production of abnormal mitotic spindles.Accordingly, the invention extends, in a second aspect, to the use of acompound of general formula I as an antimitotic compound.

The compound may be used in vivo or in vitro as an antimitotic compoundin, for example, procedures that require the blocking of cells inmitosis, such as the preparation of mitotic spreads for karyotypeanalysis and the probing of microtubule function in mitotic cells.

In a third aspect, the invention provides a novel compound of generalformula I as described herein, but excluding a single compound ofgeneral formula I wherein R₁ represents methyl, R₂ represents ahydrogen, R₇₀ represents methyl, R₇₁ represents methyl, R₇₃ representshydrogen, n represents 0, R₃ represents t-butyl, R₇₄ representshydrogen, R₆ represents methyl, R₇ represents methyl, R₇₂ representshydrogen and R₇₅ represents —CH(CH(CH₃)₂)CH.CCH₃.COOH. The excludedcompound is hemiasterlin.

Preferably, there is provided a compound of general formula I asdescribed herein, but excluding a compound in which R₁ represents amethyl group.

Preferably, there is provided a compound of general formula I asdescribed herein, but excluding a compound in which R₂ represents ahydrogen atom.

One class of preferred novel compounds comprises hemiasterlins ofgeneral formula I wherein:

-   R₁ represents a hydrogen atom;-   R₂ represents a hydrogen atom, or an alkyl group, or an acyl group;-   R₃ represents a hydrogen atom, or an optionally substituted alkyl    group;-   n represents O;-   R₇₀ and R₇₁ independently represent a hydrogen atom or optionally    substituted alkyl group, but preferably each represent a methyl    group;-   R₇₂, R₇₃ and R₇₄ represent hydrogen atoms;-   R₇ represents a hydrogen atom or an alkyl group;-   R₆ represents a hydrogen atom, or an optionally substituted alkyl    group, or a methylene group bonded to the indole moiety thereby to    form a tricyclic moiety;-   R₇₅ represents a group of general formula III described above    wherein R₄ represents a hydrogen atom, or an optionally substituted    alkyl group; R₅ represents a hydrogen atom or an alkyl group; R₇₆    and R₇₇ represent radicals as described; and X represents a group    —OR₈ or a group —NR₉R₁₀, wherein R₈, R₉ and R₁₀ independently    represent a hydrogen atom or an optionally substituted alkyl group.

Another class of preferred novel compounds comprises hemiasterlins ofgeneral formula I wherein:

-   R₁ represents a hydrogen atom or an alkyl group;-   R₂ represents an acyl group;-   R₃ represents a hydrogen atom, or an optionally substituted alkyl    group;-   n represents O;-   R₇₀ and R₇₁ independently represent a hydrogen atom or optionally    substituted alkyl group, but preferably each represent a methyl    group;-   R₇₂, R₇₃ and R₇₄ represent hydrogen atoms;-   R₇ represents a hydrogen atom or an alkyl group;-   R₆ represents a hydrogen atom, or an optionally substituted alkyl    group, or a methylene group bonded to the indole moiety thereby to    form a tricyclic moiety;-   R₇₅ represents a group of general formula III described above    wherein R₄ represents a hydrogen atom, or an optionally substituted    alkyl group; R₅ represents a hydrogen atom or an alkyl group; R₇₆    and R₇₇ represent radicals as described; and X represents a group    —OR₈ or a group —NR₉R₁₀, wherein R₈, R₉ and R₁₀ independently    represent a hydrogen atom or an optionally substituted alkyl group.

Another class of preferred novel compounds comprises criamides ofgeneral formula I wherein:

-   R₁ represents a hydrogen atom or an alkyl group;-   R₂ represents a hydrogen atom, or an alkyl group, or an acyl group;-   R₃ represents a hydrogen atom, or an optionally substituted alkyl    group;-   n represents O;-   R₇₀ and R₇₁ independently represent a hydrogen atom or optionally    substituted alkyl group, but preferably each represent a methyl    group;-   R₇₂, R₇₃ and R₇₄ represent hydrogen atoms;-   R₆ represents a hydrogen atom, or an optionally substituted alkyl    group, or a methylene group bonded to the indole moiety thereby to    form a tricyclic moiety;-   R₇₅ represents a group of general formula III described above    wherein R₄ represents a hydrogen atom, or an optionally substituted    alkyl group; R₅ represents a hydrogen atom or an alkyl group; R₇₆    and R₇₇ represent radicals as described; and X represents a group    —NR₉R₁₀, wherein R₉ and R₁₀ independently represent a hydrogen atom    or an optionally substituted alkyl group.

In formulas I and III drawn above, the bonds drawn in wavy line are fromcarbon atoms which are, or may be, optical centres.

Preferably, in the compound of general formula I the following opticalconfigurations predominate.

In accordance with a further aspect of the present invention there isprovided a geodiamolide compound of general formula

wherein:

-   R₅₁ represents an alkyl group;-   R₅₂ represents a hydrogen atom or an alkyl group; and-   A represents a halogen atom.

Preferably, R₅₁ represents a methyl group.

Suitably, R₅₂ represents a hydrogen atom or, preferably, a methyl group.

Suitably, A represents a chlorine, bromine or, preferably, an iodineatom.

In the formula IV as drawn above the bonds shown in wavy line are fromcarbon atoms which are optical centres, except for the carbon atomscarrying moieties R₅₁ and R₅₂, when those moieties are hydrogen atoms.Preferably, the following optical configuration predominates.

Certain compounds of the general formulae I and IV as defined above maybe obtained from the marine sponge Cymbastela sp. (formerly classifiedas Pseudaxinyssa sp.); or be a derivatisation of compounds obtainedtherefrom. Derivatisation of compounds of general formula I may involvestandard acylation of the extracted compounds, optionally by standardesterification. Alternatively compounds of the general formula I and IVmay be prepared by entirely synthetic routes.

The invention extends to the use of a compound of general formula IV forthe preparation of a medicament for use in therapy.

Compounds of general formula I may be prepared by coupling amino acidmoieties A, B and C as represented below.

The coupling reactions may involve standard procedures. The amino acidmoieties A, B, C may be prepared by standard procedures or in proceduresanalogous to the procedures described in the examples hereinafter.

One general procedure for the preparation of certain compounds ofgeneral formula I is provided below.

Compounds of general formula I wherein R₇₅ represents

may be prepared by reacting a compound of general formula

with a compound of general formula

A coupling agent, for example N,N′-dicyclohexylcarbodiimide (DCC), issuitably used in the reaction. The reaction suitably comprisescontacting compounds V and VI in the presence of the coupling agent, abase such as triethanolamine (TEA) and an organic solvent, such asacetonitrile, suitably at a reduced temperature. After a period of time,an inorganic base, for example sodium hydroxide may be added and,subsequently, the temperature raised to ambient temperature andtrifluoroacetic acid (TFA) added. The desired compound of generalformula I may then be isolated by standard techniques.

Compounds of general formula VI may be prepared by reacting a compoundof general formula

wherein BOC (tert-butoxycarbonyl) is a protecting group, with an ylid offormula for example (Ph)₃═CR₅CO₂R₈. The reaction is suitably carried outin the presence of potassium carbonate in a 1:1 mixture of THF/water asdescribed in R. Lloyd (1994) Ph.D. Thesis, University of Cambridge. Theprotecting group BOC is suitably removed, when required, by reaction inTFA for about 2 hours at ambient temperature.

Compounds of general formula VII may be prepared by reacting a compoundof general formula

with a reducing agent, for example lithium aluminium hydride intetrahydrofuran.

Compounds of general formula VIII may be prepared from compounds ofgeneral formula

using the method described in Synthesis 1983, 676.

Compounds of general formula IX may be prepared from compounds ofgeneral formula

by reaction with a compound of general formula R₇I in the presence of analkali metal hydride and in THF, using the method described inCan.J.Chem. 1973, 51, 1915.

Compounds of general formula V may be prepared by reacting a compound ofgeneral formula

initially with a base, for example dilute sodium hydroxide solution,followed by acidification down to about pH6. In some circumstances, itis desirable to protect the group —NR₆R₂ from reaction and this issuitably afforded using a protecting agent such as BOC.

Compounds of general formula XI may be prepared by reacting a compoundof general formula

with a base followed by treatment with an azide compound. The azidederivative of compound XII may then be reduced to form an aminederivative which may then be treated with groups R₆₁ and/or R₂₁ in thepresence of a base, for example sodium hydride, to afford the groupR₆R₂N— in the compound of formula XI.

Compounds of general formula XII may be prepared by coupling compoundsof general formula

using a coupling agent, such as DCC, a base such as TEA and in anorganic solvent such as acetonitrile suitably at about 0° C.

Compounds of general formula XIII may be prepared from a compound ofgeneral formula

by processes well known to skilled persons in the art.

Compounds of general formula XIV and XV may be prepared by processeswell known to skilled persons in the art.

Compounds of general formula I wherein R₇₅ represents

may be prepared by reacting a compound of general formula I as describedabove with an amine of general formula R₉R₁₀NH. The reaction is suitablycarried out in the presence of a coupling agent such as DCC, a base suchas TEA and in an organic solvent, such as acetonitrile, suitably at areduced temperature. In certain circumstances, the group —NR₆R₂ may needprotecting from undesired reactions and this is suitably afforded usinga protecting agent such as BOC.

In accordance with a further aspect of the present invention there isprovided a method of obtaining a compound of general formula I byextraction of a compound of general formula I from Cymbastela sp.,including the steps of separation and purification; and optionallyderivatising said compound to derive a further compound of generalformula I.

Derivatisation of a compound of general formula I may include acylationand/or esterification steps. Esterification and/or acylation steps maybe undertaken under standard conditions.

In accordance with a further aspect of the present invention there isprovided a method of obtaining a compound of general formula IV byextraction of a compound of general formula IV from Cymbastela sp.including the steps of separation and purification; and optionallyderivatising said compound to derive a further compound of generalformula IV.

Compounds of the general formula I and IV are biologically active. Theinvention further relates to the biological use of a compound of generalformula I or IV. Compounds of general formula I or IV may havepesticidal, for example insecticidal activity. Preferably, however, theuse is in the veterinary or, most preferably, the pharmaceutical field.

The compounds described herein may in particular have utility asantibacterial and/or antiviral agents, and/or, especially, as cytotoxicagents.

The invention further provides the use of any compound of generalformula I or IV for the manufacture of a medicament for use in thetreatment of cancer or a tumor in a mammal.

In using a compound of general formula I or IV as described in anystatement herein, the compound is preferably administered to a patientin a pharmaceutical or veterinary composition comprising also apharmaceutically or veterinarily acceptable carrier, and optionally, oneor more other biologically active ingredients. Such compositions may bein any form used for administering pharmaceuticals, for example any formsuitable for oral, topical, vaginal, parenteral, rectal and inhalatoryapplication. The compositions may be provided in discrete dose units.The carriers may be particulate, with the compositions being, forexample, tablets or powders, or liquid, with the compositions being, forexample, oral syrups or injectable liquids, or gaseous, for inhalatoryapplication.

For oral administration an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Colouring and/orflavouring agents may be present. A coating shell may be employed. Forrectal administration oleaginous bases may be employed, for examplelanolin or cocoa butter. For an injectable formulation buffers,stabilisers and isotoic agents may be included.

The dosage of the compounds of general formula I and IV may depend uponthe weight and physical condition of the patient; on the severity andlongevity of the illness; and on the particular form of the activeingredient, the manner of administration and the composition employed. Adaily dose of from about 0.001 to about 100 mg/kg of body weight takensingly or in separate doses of up to 6 times a day, or by continuousinfusion, embraces the effective amounts most typically required. Apreferred range is about 0.01 to about 50 mg/kg of body weight, per day,most preferably about 0.1 to about 30 mg/kg of body weight, per day.

It is to be understood that use of a compound of general formula I or IVin chemotherapy can involve such a compound being bound to an agent, forexample a monoclonal or polyclonal antibody, a protein or a liposome,which assists the delivery of the said compound to tumour cells.

Therefore, the invention relates further to a pharmaceutical andveterinary composition comprising an. effective amount of a compound offormula I or IV, in association with a carrier.

The invention will now be further described, by way of example withreference to FIGS. 1 and 2 which are graphs relating to antimitoticactivity.

Procedure 1:—Isolation of Naturally Occurring Compounds

Specimens of Cymbastela sp. were collected by hand using SCUBA on reefsoff Mandang, Papua, New Guinea. Freshly collected sponge was frozen onsite and transported to Vancouver, Canada, over dry ice. The spongeswere identified by a leading sponge taxonomy expert Professor R. vanSoest. A voucher sample was been deposited at the Institut voorSystematrek en Populatiebiologie-Zoologisch Museum, University ofAmsterdam.

The thawed sponge (260 g dry wt) was extracted exhaustively with asolution of CH₂Cl₂/MeOH (1:1). Evaporation of the organic extract invacuo gave an aqueous suspension. MeOH was added to give a 9:1 MeOH:H₂Osolution (1 l), which was extracted with hexanes (4×250 ml). The hexaneextracts were combined and concentrated in vacuo to yield an orange oil.Water was added to the MeOH solution to give a 1:1 MeOH/H₂O solution,which was extracted with CHCl₃ (4×250 ml). The combined CHCl₃ layerswere concentrated in vacuo to yield an orange oil (3.5 g). Repeated sizeexclusion chromatography on Sephadex LH-20 eluting with MeOH yielded anumber of crude geodiamolides and hemiasterlins. Pure geodiamolide G(Compound 1 below) (2 mg, 0.0007% dry wt) was obtained viareversed-phase HPLC (MeOH/H₂O 60:40). Reversed-phase isocratic HPLC(0.05% TFA:MeOH 1:1) afforded hemiasterlin A (Compound 2 below −32 mg,0.012% dry wt) and hemiasterlin B (Compound 3 below −1 mg, 0.0004% drywt). These compounds are novel. The reversed-phase isocratic HPLC usingTFA and MeOH also yielded the known compound hemiasterlin (Compound Abelow −40 mg, 0.015% dry wt). This was used to prepare a novel acylatedand esterified compound described later.

-   Geodiamolide G (compound 1): colourless glass; IR (neat) 3313, 2977,    2933, 1732, 1675, 1635, 1505, 1455, 1417, 1377, 1285, 1217, 1102,    1083, 1052, 952, 827, 754 cm⁻¹; NMR data, Table 1 below; HREIMS, M⁺    m/z 655.1760 (C₂₈H₃₈N₃O₇I ΔM 0.6 mmu).-   Hemiasterlin A (Compound 2): white solid [α]D=−45° (c 0.25, MeOH);    UV (MeOH) λmax (ε) 218 (23,400), 280 nm (2,800); IR (neat) 3418,    2966, 1689, 1680, 1643 cm⁻¹; NMR data, Table 2 below; HRFABMS, MH⁺    m/z 513.3471 (C₂₉H₄₅O₄N₄ ΔM 3.0 mmu).-   Hemiasterlin B (Compound 3): white solid; CD(MeOH) (Θ)₂₂₆ 10,800;    NMR data, Table 2 below; HRFABMS, MH⁺ m/z 499.3319 (C₂₈H₄₃O₄N₄ ΔM    3.4 mmu).-   Hemiasterlin (Compound A): white solid; [α]D=−77° (c 0.07, MeOH); UV    (MeOH) λmax (ε) 216 (15,400), 273 nm (1,600); IR (neat) 3412, 2962,    1650, 1635 cm⁻¹; NMR data, Table 2 below; HRFABMS, MH⁺ m/z 527.3594    (C₃₀H₄₇O₄N₄ ΔM −0.35 mmu).

Product identification, including assignment of stereochemicalconfigurations, was achieved by a range of techniques, including NMR,mass spectroscopy and optical rotation measurements, withcross-reference to analyses reported in the literature, for compoundsalready known. In the case of compounds 2 and 3 CD and chemicaldegradation analyses were carried out to assist the stereochemicaldetermination.

TABLE 1 NMR Data for Geodiamolide G (Compound 1). Recorded in CDCl₃ at500 MHz. Carbon δ ¹³C^(a) δ ¹H COSY HMBC^(b)  1 174.5H3.H3′.H14.H22.NH(14)  2 41.0 2.46.m H3.H3′.H22 H3′.H22  3 37.72.55.ddJ=12.3.3.7Hz H2.H3′ H22.H23.H23′  3′ 2.14.tJ=11.9Hz H2.H3  4143.6 H3.H23′  5 205.1 H3′.H23.H23′.H24  6 36.2 2.95 H7′.H24 H24  7 38.81.82.dddJ=14.6.9.5.2.8Hz H7′.H8 H24.H25  7′ 1.61.dddJ=14.6.10.9.2.8HzH6.H7  8 69.7 5.11.m H7.H25 H25  9 170.6 H10.H26 10 49.14.51.daJ=7.3.7.2Hz H26.NH(10) H26 NH(10) 6.35.dJ=7.3Hz H10 11 168.9H12.H15′ 12 57.1 5.06.ddJ=7.9.8.9Hz H15.H15 H15.H15′.H27 13 174.4H12.H14.H27.H28.NH(14) 14 45.1 4.72.daJ=7.0.6.9Hz H28.NH(14) H28 NH(14)6.19.dJ=7.0Hz H14 15 33.2 3.12.ddJ=14.6.7.9Hz H12.H15′ H12.H17.H21 15′2.90.ddJ14.6.8.9Hz H12.H15 16 130.3 H15.H15′.H20 17 138.2 7.45.dJ=1.4HzH21 H15.H15′.H21 18 85.2 H17.H20 19 154.5 H17.H21 20 115.6 6.88.dJ=8.2HzH21 21 130.6 7.04.ddJ=8.2.1.4Hz H17.H20 H17 22 17.9 1.15.dJ=6.4Hz H2 23127.8 5.90.s H3′ 23′ 5.78.s 24 17.8 1.09.dJ=7.1Hz H6 25 20.81.28.dJ=6.3Hz H8 26 18.3 1.32.dJ=7.2Hz H10 H10 27 30.7 2.97.s H12 2819.2 1.04.dJ=6.9Hz H14 ^(a)Obtained from HMQC and HMBC spectra only.^(b)Proton resonances that are correlated to the carbon resonance in theδ ¹³C column.

TABLE 2 NMR Data for the hemiasterlins, Compounds 2, 3 and A. Recordedin DMSO-d₆ at 500 MHz. Hemiasterlin Hemiasterlin A Hemiasterlin B Carbonδ ¹³C δ ¹H δ ¹³C δ ¹H HMBC^(a) δ ¹³C^(b) δ ¹H HMBC^(a)  1-N 10.93.s10.88.s  2 128.7 7.16.s 122.9 7.11.s H1 122.9 7.06.s  3 116.5 120.4H2.5.6.14.15 119.0 H14.15  4 125.0 124.8 H1.2.8 124.8 H2.8  5 120.68.09.d.J=8Hz 120.2 7.80.d.J=8Hz 120.0 7.98.d.J=7.8Hz  6 121.17.07.t.J=8Hz 120.7 7.06.t.J=8Hz 120.4 7.05.t.J=7.8Hz  7 118.47.20.t.J=8Hz 118.1 6.96.t.J=8Hz H8 117.8 6.95.t.J=7.8Hz H8  8 110.07.44.d.J=8Hz 111.8 7.35.d.J=8Hz 111.4 7.38.d.J=7.8Hz H7  9 137.7 137.3H1.2.5.6 137.2 H2 10 37.5 37.9 H11.14.15 37.5 H14.15 11 67.54.44.d.J=6Hz 71.7 3.47.s H14.15.17 69.3 3.47.bs 12 166.0 171.2 H11 1332.4 3.75.s 14 27.0 1.41.s 27.5 1.41.s H15 27.3 1.38.s H15 15 22.51.38.s 23.2 1.37.s H11.14 22.5 1.34.s H14 16-N 7.38.bs 17 33.4 2.24.s35.2 1.92.s H11 35.0 1.93.s 18-N 8.87.s 7.84.bd.J=9Hz 19 56.24.84.d.J=8Hz 53.8 4.79.d.J=9Hz 53.8 4.58.t.J=8Hz H22.23 20 170.1 170.9H19.30 171.0 H30 21 34.6 34.7 H19.22.23.24 30.0 2.11.m H19.22 22 26.30.99.s 26.2 0.93.s H19.23.24 0.84.d.J=6.3Hz 23 26.3 0.99.s 26.2 0.93.sH19.22.24 0.89.d.J=6.3Hz 24 26.3 0.99.s 26.2 0.93.s H19.22.23 26 55.64.93.t.J=10Hz 55.9 4.91.t.J=9Hz H30.32.33 55.8 4.87.t.J=10Hz H30.32.3327 138.3 6.66.d.J=10Hz 138.2 6.63.d.J=9Hz H26.34 137.7 6.63.d.J=8.8HzH26.34 28 131.6 131.8 H26.34 131.5 H34 29 168.5 168.6 H27.34 168.5 H3430 31.1 3.03.s 30.9 2.97.s H26 30.0 2.98.s 31 28.7 2.01.m 28.8 1.96.mH26.32.33 28.7 2.08.m H32.26 32 19.3 0.80.d.J=7Hz 19.3 0.77.d.J=6.5HzH33 0.74.d.J=6.1Hz 33 18.9 0.78.d.J=7Hz 18.7 0.70.d.J=6.5Hz H320.80.d.J=6.1Hz 34 13.5 1.80.s 13.5 1.77.s H27 13.0 1.75.s H27 ^(a)Protonresonances that are correlated to the carbon resonance in the δ¹³Ccolumn. ^(b)Obtained from HMQC and HMBC correlations.

In another example, specimens of Cymbasyela sp. were collected by handusing SCUBA on the reefs of Madang, Papua, New Guinea. Fresh sponge wasfrozen on site and transported to Vancouver, Canada, over dry ice. Thefreeze-dried sponge (157 g dry wt.) was extracted sequentially withhexane, carbon tetrachloride, chloroform and methanol (3×8 liters). Theextracts were concentrated in vacuo to yield 6 g, 0.76 g, 1.24 g and 1.1g respectively for each solvent. Repeated size exclusion chromatographyof the chloroform extract, on Sephadex LH-20 with methanol, yielded amixture of crude hemiasterlins and criamides. Reversed phase HPLC, 50:500.05% TFA:MeOH afforded pure Hemiasterlin (60 mg, 0.038% dry wt.)(Compound A above); Hemiasterlin-A (55 mg, 0.035% dry wt.) (Compound 2above); Hemiasterlin-B (3 mg, 0.0019% dry weight) (Compound 3 above);Criamide-A (2 mg, 0.0013% dry wt.) (Compound 6 below); Criamide-B (2 mg,0.0013% dry wt.) (Compound 7 below).

Product identification of compounds 6 and 7, including assignment ofstereochemical configurations, was achieved by a range of techniques asdescribed above for the other examples. NMR data is provided in Table 3below.

TABLE 3 NMR Data for Criamide B (Compound 7) and Criamide A (Compound 6)Criamide B Criamide A Carbon δ ¹³C (ppm) δ ¹H (ppm) COSY HMBC δ ¹H (ppm)COSY no. (125 MHz) (500 MHz) (500 MHz) (500 MHz) (500 MHz) (500 MHz) 1-N 11.14.s  2 124.2 7.16.d.J=2.3 7.17.s  3 117.1 H1.2.14.15  4 137.7H5.7  5 120.2 8.08.d.J=7.3 H6 H7 8.10.d.J=7.9 H6  6 118.0 7.03.t.J=7.3H5.7 H8 7.08.t.J=7.0 H5.7  7 120.9 7.12.t.J=7.3 H6.8 H5 7.21.t.J=8.2H6.8  8 112.0 7.41.d.J=8.0 H7 H6 7.46.d.J=8.5 H7  9 124.7 H1.2.6 10 37.6H14.15 11 67.6 4.45.d.J=9.0 H16-A H14.15 4.44.d.J=9.2 H16-A 12 165.8 H1913 3.75.s 14 27.2 1.42.s H15 1.42.s 15 22.0 1.38.s H14 1.38.s 16 A7.30.bs H16-B.11.17 7.35.bs H16-B.11.17 B 8.77.bs H16-A.17 8.84.bsH16-A.17 17 33.5 2.23.t.J=4.8 H16A.16B 2.24.bs H16A.16B 18-N8.86.d.J=9.2 H19 8.87.d.J=6.1 H19 19 55.5 4.88.d.J=8.2 H18 H22.23.244.88.d.J=8.5 H18.H21 20 170.1 H19.26.30 21 34.9 H22.23.24 22 26.3 1.0.sH19.23.24 1.00.s 23 26.3 1.0.s H19.22.24 1.00.s 24 26.3 1.0.s H19.22.231.00.s 26 55.9 4.98.t.J=9.8 H27.31 H30.32.33 4.98.t.J=9.8 H27.31 27132.4 6.30.d.J=9.0 H26 H26.34 6.30.d.J=8.5 H26 28 135.4 H26.34 29 168.7H27.29.34 30 30.9 3.04.s H26 3.01.s 31 28.7 2.01.m H26.32.33 H32.331.94.m H26.32.33 32 19.5 0.83.d.J=6.5 H31 H33 0.83.d.J=7.7 H31 33 19.20.78.d.J=6.5 H31 H32 0.77.d.J=7.7 H31 34 14.2 1.82.s H27 1.82.s 35-N8.02.d.J=7.6 H36 8.03.d.J=7.6 H36 36 51.8 4.27.m H35.38-A.38-B4.26.dd.J=4.0.9.0 H35.38-A.38-B 38 27.7 A 1.81.m H36.38-B A 1.79.mH36.38-B B 1.67.m H38-A.39 B 1.69.m H38-A.39 39 24.6 1.51.m H38-B.401.49.m H38-B.40 40 40.0 3.10.dd.J=5.9.7.1 H39.41 3.09.dd.J=6.0.7.0H39.41 41-N 7.53.t.J=5.9 H40 7.59.t.J=5.0 H40Procedure 2: Derivatisation of Naturally Occurring Compounds

The following compounds 4 and 5, were respectively prepared fromcompounds A and 2 above.

They were prepared by firstly converting compounds A and 2 to theirmethyl esters, then by acylating the esters.

To prepare the methyl esters of compounds A and 2, diazomethane wasprepared in the standard fashion from1-methyl-3-nitro-1-nitrosoguanidine (MNNG) using an AldrichMNNG-diazomethane kit. The resulting yellow diazomethane solution (3 mlether) was added to 1 mg of peptide A or 2, dissolved in 3 ml ofchloroform. The reaction mixture was left at ambient temperature for onehour then the solvents were removed in vacuo.

To then acylate the esterified peptides, approximately 1 mg of eachesterified peptide was stirred overnight under an argon atmosphere withfreshly distilled (from NaOH) pyridine and acetic anhydride (1.0 mleach). Excess reagents were removed in vacuo to yield the pure N-acetylpeptide esters, compounds 4 and 5, which are characterised as follows.

-   Hemiasterlin, N-acetyl methyl ester (Compound 4): white solid;    CD(MeOH) (Θ)₂₃₁+13,300; ¹H NMR CD₂CL₂, 500 MHz) δ 0.44 (s), 0.84 (d,    J=6.6 Hz), 0.87 (d, J=6.6 Hz), 1.39 (s), 1.59 (s), 1.86 (s), 2.04    (m), 2.16 (s), 2.93 (s), 3.15 (s), 3.70 (s), 3.76 (s), 4.41 (d, J=9    Hz), 5.03 (t, J=9 Hz), 6.18 (d, J=8 Hz), 6.39 (s), 6.64 (d, J=8 Hz),    7.10 (s), 7.15 (t, J=8 Hz), 7.24 (t, J=8 Hz), 7.32 (d, J=8 Hz), 8.29    (d, J=9 Hz); EIHRMS, M⁺ m/z 582.3796 (C₃₃H₅₀N₄O₅ ΔM 1.5 mmu).-   Hemiasterlin A, N-acetyl methyl ester (Compound 5): white solid;    CD(MeOH) (Θ)₂₃₁+10,400; ¹H NMR CD₂Cl₂, 500 MHz) δ 0.48 (s), 0.83 (d,    J=6.6 Hz), 0.88 (d, J=6.6 Hz), 1.40 (s) 1.54.(s), 1.85 (s), 1.98    (m), 2.16 (s), 2.93 (s), 3.15 (s), 3.70 (s), 4.48 (d, J=10.1 Hz),    5.02 (t, J=10 Hz), 6.19 (d, J=9 Hz), 6.37 (s), 6.65 (d, J=10.5 Hz),    7.17 (t, J=7 Hz), 7.20 (t, J=7 Hz), 7.23 (s), 7.40 (d, J=7 Hz), 8.30    (d, J=10 Hz), 8.31 (s); EIHRMS, M⁺ m/z 568.3626 (C₃₂H₄₈N₄O₅ ΔM 0.1    mmu).    Procedure 3: Totally Synthetic Method

The process for preparing compounds described herein by a totallysynthetic method involves, in general terms, the coupling of aminoacids. Thus, the preparation of the compound

involves coupling amino acids corresponding to units A, B and C.Criamide compounds which include an additional moiety D coupled to end Cof the compound shown above can be prepared by coupling an amino acidcorresponding to the desired moiety D to the peptide A-B-C.

The preparations of amino acids A and C are described below. Amino acidB is commercially available.

1. Preparation of Amino Acid C (N-methylhomo Vinylogous Valine EthylEster)

Scheme 1 below provides a summary of the procedures describedhereinafter.

(a) Preparation of N-Boc,N-Me-L-valine (1)

N-Boc-L-valine (5 g; 23 mmol) and methyl iodide (1.59 ml; 25.3 mmol)were dissolved in THF (65 ml) and the solution was cooled to 0° C. underargon. Sodium hydride dispersion (2.03 g; 50.6 mmol) was addedcautiously with gentle stirring. The suspension was stirred at roomtemperature for 16 h. Ethyl acetate (30 ml) was then added (to consumethe sodium hydroxide formed from the excess sodium hydride), followed bywater, drop wise, to destroy the excess sodium hydride. The solution wasevaporated to dryness, and the oily residue partitioned between ether(25 ml) and water (50 ml). The ether layer was washed with 5% aqueousNaHCO₃ (25 ml), and the combined aqueous extracts acidified to pH 3 withaqueous citric acid. The product was extracted into ethyl acetate (3×25ml), the extract washed with water (2×25 ml), 5% aqueous sodiumthiosulfate (2×25 ml; to remove iodine), and water (2×25 ml), dried overMgSO₄ and evaporated to give a yellow oil. The procedure was repeated toimprove the overall yield. Final yield was 3.53 g; 70.6%. ¹H nmr (CDCl₃;400 MHz) δ 0.86 (d, 3H, J=7 Hz), 0.97 (d, 3H, J=7 Hz),1.41 (s, 9H),2.17(bs, 1H),2.80 & 2.82 (2 s, 3H), 4.05 (d, 0.5H, J=10 Hz), 4.26 (d, 0.5H,J=10 Hz), 10.8 (bs, 1H).

(b) Preparation of N-Boc,N-Me-L-valine-N-Me,N-Ome (Weinreb amide)(2)

N-Boc,N-Me-L-valine (3.2 g; 13.9 mmol), N,O-dimethylhydoxylaminehydrochloride (1.5 g; 15 mmol) and triethyl amine (2.1 ml; 30 mmol) weredissolved in CH₂Cl₂ (30 ml) and the solution cooled to −10° C. underargon. Dicyclohexylcarbimide (3.1 g; 15 mmol) was dissolved in 15 ml ofCH₂Cl₂ and added drop wise to the reaction mixture over 10 minutes. Thesolution was stirred for an additional 15 minutes at −10° C. and thenfor 1 h at room temperature at which time it was filtered and the excesssolvents removed in vacuo. The oil was dissolved in EtOAc (50 ml) andwashed with 5% HCl (2×25 ml), water (2×25 ml), 5% NaHCO₃ (2×25 ml), andwater (2×25 ml), dried over MgSO₄ and evaporated to yield a yellow oil(3.4 g; 85% yield).

¹H nmr (CDCl3; 400 MHz) δ (0.82 (t, 6H J=7 Hz), 1.38 (s, ˜3 H), 1.41 (s,˜6H), 2.16 (m, 1H), 2.73 (s, ˜1H), 2.76 (s, ˜2H), 3.13 (s, 3H), 3.62 (s,˜1H), 3.65 (s, ˜2H). Doubling of peaks caused by rotamers around theN-Boc group.

(c) Preparation of N-Boc, N-Me-L-valine Aldehyde (3)

Weinreb amide (226 mg; 0.78 mmol) was dissolved in THF (8 ml) and cooledto −78° C, then added drop wise to a dispersion of LiAlH₄ (35 mg; 0.86mmol) in THF at −78° C. The reaction was stirred for 0.5 h at which timeit was quenched with Na₂SO₄.10H₂O (251 mg; 0.78 mmol) and allowed towarm to room temperature. The solution was filtered through celite andthe excess solvents removed in vacuo to yield a colourless oil. Normalphase silica gel chromatography, eluting with 1:6 ethyl acetate:hexanes, afforded pure N-Boc,N-Me-L-valine aldehyde as a clear oil (116mg; 68%).

¹H nmr (CDCl₃; 400 MHz) δ 0.84 (d, 3H, J=7 Hz), 1.03 (d, 3H, J=7 Hz),1.40 (s,9H), 2.20 (bs, 1H), 2.72 (s,2H), 2.84 (s,1H) 3.55 (d, 0.5 H,J=10 Hz), 4.02 (d,0.5H, J=10 Hz), 9.58 (bs, 1H).

(d) Preparation of N-Boc-MHVV-OEt (4)

N-Boc,N-Me-valine aldehyde (120 mg; 0.56 mmol) was dissolved in a 1:1mixture of THF:H₂O (6 ml) and the (carbethoxyethylidene)triphenylphosphane (222 mg; 6.2 mmol) was added and thereaction stirred at room temperature for 4 h. Normal phase silica gelchromatography, eluting with 1:6 ethyl acetate: hexanes, afforded theN-Boc-homo vinylogous valine ethyl ester as a clear oil (212 mg; 71%).

¹H nmr (CDCl3; 400 MHz) δ 0.82 (d, 3H, J=7 Hz), 0.87 (d, 3H, J=7 Hz),1.27 (t,3H, J=7 Hz), 1.42 (s, 9H), 1.85 (m, 1H), 1.87 (s, 3H), 2.69 (bs,3H) 4.17 (q, 2H, 7 Hz), 4.28 (bs, 0.5H), 4.53 (bs, 0.5H), 6.62 (d, 1H,J=9 Hz).

(e) Preparation of MHVV-OEt (5)

N-Boc-MHVV-OEt (200 mg; 67 mmol) was dissolved in 1 ml of 1:1 CH₂Cl₂:trifluoroacetic acid mixture and stirred under argon for 0.5 h. Excesssolvents were removed in vacuo and the oily residue was twiceredissolved in CH₂Cl₂ (25 ml) and concentrated to remove any traces ofTFA. The final product was a white amorphous solid (207 mg, 99%).

¹H nmr (CDCl3; 400 MHz) δ 0.82 (t, 6H, J=7 Hz), 1.27 (t, 3H, J=7 Hz),1.42 (s, 9H), 1.85 (m, 1H), 1.87 (s, 3H), 2.69 (bs, 3H), 3.9 (m, 1H),4.17 (q, 2H, 7 Hz), 6.62 (d, 1H, J=9 Hz) 8.1 (bs, 0.5H), 8.3 (bs, 0.5H),12.9 (bs, 1H).

2. Preparation of Amino-acid A (N-Boc-tetramethyltryptophan Derivative)

Scheme 2 below provides a summary of the procedures describedhereinafter for the preparation of amino acid A and derivatives whichare described herein:

(a) Preparation of Methyl Ester (7)

To a stirred suspension of indol-3-acetic acid (6) (1.07 g, 6.11 mmol)in ether (20 ml) at room temperature was added an ethereal solution ofdiazomethane drop wise until the yellow colour of the diazomethanepersisted in the reaction mixture, and tlc analysis showed completeconsumption of starting material. Excess diazomethane was removed undera stream of argon and the remaining solvent removed in vacuo. The crudeoil thus obtained was purified by flash column chromatography (50% etherin pet. ether), to afford methyl ester, (7) as an off-white solid (1.16g, 100%).

Mp: 47-48° C. IR (CHCl₃ soln): 3409 (s, NH), 1729 (s, C=0), 1621 (w,C═C). ¹H NMR (400 MHz, CDCl₃) δ: 3.71 (3H,s, OCH ₃), 3.79 (2H,s, CH₂CO₂CH₃), 7.03 (1H, s, ═CHNH), 7.15 (1H,t,J=7.8 Hz, ArH), 7.20(1H,t,J=7.8 Hz, ArH), 7.31 (1H,d,J=7.8 Hz, ArH), 7.63 (1H,d,J=7.8 Hz,ArH), 8.12 (1H,br,s,NH). ¹³C NMR (75.3 MHz, CDCl3) δ: 172.6, 136.0,127.1, 123.1 122.1, 119.6, 118.7, 111.2, 108.1, 51.9, 31.9. LRMS (EI):189 (M⁺, 25%) , 130 (100%). HRMS (EI) Calcd. for C₁₁H₁₁O₂N: 189.07898;found: 189.07866. Anal. Calcd. for: C₁₁H₁₁O₂N: C, 69.83; H, 5.86; N,7.40. Found: C, 69.47; H, 5.91; N, 7.50

(b) Preparation of Di Methyl Ester (8)

To a stirred, cooled (−78° C.) suspension of potassiumbis(trimethylsilyl) amide (4.90 g 24.6 mmol) in dry THF (100 ml) underargon was added a solution of methyl ester (7) (1.57 g, 8.31 mmol) inTHF (30 ml+20 ml washings) via cannula. The reaction mixture was warmedto 0° C. and stirred for two hours before recoiling to −78° C. Freshlydistilled methyl iodide (5.2 ml, 82.8 mmol) was added, the mixtureallowed to warm to 0° C. and stirring continued for three hours or untiltlc analysis showed complete reaction. The reaction was quenched withwater (100 ml) and then extracted with ether (3×100 ml), the combinedorganic extracts were washed with brine (100 ml), dried with magnesiumsulfate and concentrated in vacuo. The resulting crude oil was purifiedby flash column chromatography (25% ether in pet.ether) to afford methylester (8) as a viscous pale yellow oil (1.61 g, 89%).

IR (neat): 1734(s, C═O), 1615, 1550 (w, C═C). ¹H NMR (400 MHz, CDCl₃) δ:1.61 (3H, d,J=7.1 Hz, CH(CH ₃)), 3.67 (3H, s, NCH ₃), 3.75 (3H,s, OCH₃), 4.01 (1H,q,J=7.1 Hz, CH(CH₃)CO₂CH₃), 7.00 (1H, s ═CHNCH₃), 7.13(1H,t,J=7.8 Hz, ArH), 7.24 (1H,t,J=7.8 Hz, ArH), 7.30 (1H,d,J=7.8 Hz,ArH), 7.68 (1H,d,J=7.8 Hz, ArH). ¹³C NMR (75.3 MHz, CDCl₃) δ: 175.6,136.9, 126.7, 126.2, 121.7, 119.2, 119.0, 113.9, 109.2, 51.9, 36.7,32.7, 18.0. LRMS (EI): 217 (M⁺, 18%), 158 (100%). HRMS (EI) Calcd. forC₁₃H₁₅O₂N: 217.11028; found: 217.11013. Anal. Calcd. for: C₁₃H₁₅O₂N: C,71.87; H, 6.96; N, 6.45. Found: C, 71.52; H,6.80; N, 6.26.

(c) Preparation of Tri Methyl Ester (9)

To a stirred, cooled (−78° C.) suspension of potassiumbis(trimethylsilyl)amide (2.90 g, 14.5 mmol) in dry THF (60 ml) underargon was added a solution of methyl ester (8) (1.25 g, 5.76 mmol) inTHF (30 ml+20 ml washings) via cannula. The reaction mixture was warmedto 0° C. and stirred for two hours before re-coiling to −78° C. Freshlydistilled methyl iodide (3.5 ml, 57.6 mmol), was added, the mixtureallowed to warm to 0° C. and stirring continued for three hours or untiltlc analysis showed complete reaction. The reaction was quenched withwater (60 ml) and then extracted with ether (3×75 ml), the combinedorganic extracts were washed with brine (75 ml), dried with magnesiumsulfate and concentrated in vacuo. The resulting crude oil was purifiedby flash column chromatography (20% ether in pet.ether) to afford themethyl ester (9) as an off-white solid (1.22 g 92%).

Mp: 99-101° C. IR (CHCl₃ soln): 1727 (s, C═O), 1618, 1550 (w, C═C). ¹HNMR (400 MHz, CDCl₃) δ: 1.70 (6H,S,CCH ₃(CH ₃)), 3.64 (3H,s,NCH ₃), 3.75(3H,s,OCH ₃), 6.94 (1H,s ═CHNH), 7.10 (1H,t,J=7.9 Hz ArH), 7.22(1H,t,J=7.9 Hz, ArH), 7.29 (1H,d,J=7.8 Hz,ArH), 7.64 (1H,d,J=7.8 Hz,ArH). ¹³C NMR (75.3 MHz, CDCl₃) δ: 177.6, 137.4, 125.9, 125.2, 121.5,120.2, 119.1, 119.0, 109.3, 52.1, 41.9, 32.7, 26.3. LRMS (EI): 231 (M⁺,15%), 172 (100%). HRMS (EI) Calcd. for C₁₄H₁₇O₂N: 231.12593; found:231.12572. Anal. Calcd for: C₁₄H₁₇O₂N: C, 72.70; H, 7.41; N, 6.06.Found: C, 72.83; H, 7.44; N, 6.04.

(d) Preparation of Alcohol (10)

To a stirred colled (−78° C.) solution of methyl ester (9) (1.38 g, 5.97mmol) in dry ether (70 ml) under an argon atmosphere was added DIBAL(14.9 ml, 1.0M in hexanes, 14.9 mmol). The resulting solution wasallowed to warm to 0° C. and stirring was continued for three hours. Thereaction was quenched by addition of water (30 ml), allowed to warm toroom temperature whereupon Rochelles salt (30 ml) was added. The organiclayer was separated and the aqueous layer was extracted with ether (2×50ml). The combined organic extracts were washed with brine (50 ml), driedwith magnesium sulfate and concentrated in vacuo. The crude mixture waspurified by flash column chromatography (50% ether in pet.ether) toafford the alcohol (10) as a white solid (1.14 g, 94%).

Mp: 80-82° C. IR (CHCl₃ soln): 3400 (br, s, OH), 1614, 1545 (w, C═C). ¹HNMR (400 MHz, CDCl₃) δ: 1.48 (6H,s,CCH ₃(CH ₃)), 3.75 (3H,s,NCH ₃), 3.79(2H,s,CH ₂OH), 6.90 (1H,s ═CHNH), 7.11 (1H,t,J=7.9 Hz ArH), 7.22(1H,t,J=7.9 Hz, ArH), 7.32 (1H,d,J=7.8 Hz,ArH), 7.78 (1H,d,J=7.8 Hz,ArH). ¹³C NMR (75.3 MHz, CDCl₃) δ: 137.9, 127.1, 126.1, 121.5, 121.0,119.4, 118.8, 109.6, 71.6, 37.7, 32.7, 25.5. LRMS (EI): 203 (M⁺, 17%),172 (100%). HRMS (EI) Calcd. for C₁₃H₁₇ON: 203.13101; found: 203.13052.Anal. Calcd for: C₁₃H₁₇ON: C, 76.81; H, 8.43; N, 6.89. Found: C, 76.89;H, 8.43; N, 6.70.

(e) Preparation of Aldehyde (11)

To a mixture of alcohol (10) (362 mg, 1.78 mmol) 4-methylmorpholineN-oxide (375 mg, 3.21 mmol) and 4A powdered molecular sieves (400 mg) indry dichloromethane (12 ml) under an argon atmosphere at roomtemperature was added solid TPAP (35 mg, 0.0996 mmol) in one portion.The resulting black mixture was stirred at the same temperature forthree hours, then filtered through celite to remove the molecular sievesand the filtrate concentrated in vacuo. The black oil was purified byflash column chromatography (20% ether in pet.ether) to afford thealdehyde (11) as an off-white solid (314 mg, 88%).

Mp: 61-63° C. IR (CHCl₃ soln): 1718 (s, C═O), 1610, 1542 (w, C═C). ¹HNMR (400 MHz, CDCl₃) δ: 1.58 (6H,s,CCH ₃(CH ₃)), 3.70 (3H,s,NCH ₃), 6.96(1H,s, ═CHNH), 7.10 (1H,dt,J=0.9, 8.0 Hz,ArH), 7.24 (1H,dt,J=0.9, 8.0 HzArH), 7.32 (1H,dd,J=0.9, 8.0 Hz, ArH), 7.56 (1H,dd,J=0.9, 8.0 Hz,ArH),9.49 (1H,s, CHO). ¹³C NMR (75.3 MHz, CDCl₃) δ: 202.2, 137.6, 126.6,126.1, 121.8, 120.1, 119.3, 115.0, 109.5, 46.5, 32.8, 21.9. LRMS (EI):201 (M⁺, 13%), 172 (100%). HRMS (EI) Calcd. for C₁₃ H₁₅ON: 201.1153;found: 201.11473. Anal. Calcd for: C₁₃ H₁₅ON: C, 77.58; H, 7.51; N,6.96. Found: C, 77.42; H, 7.58; N, 6.83.

(f) Preparation of Enol Ether (12)

To a stirred suspension of methoxymethyltriphenylphosphonium chloride(3.03 g, 8.84 mmol) in dry THF (40 ml) under an argon atmosphere at roomtemperature (water bath) was added potassium tert-butoxide (991 mg, 8.82mmol) as a solid in one portion. The reaction mixture immediately turneda deep red colour, the water bath was removed and stirring continued forone and a half hours at room temperature. Aldehyde (11) (828 mg, 4.12mmol) was added via cannula in THF (10 ml+5 ml washings), and stirringcontinued for a further two hours. The reaction mixture was diluted withwater (30 ml) and extracted with ether (3×40 ml). The combined organicextracts were washed with brine (60 ml), dried with magnesium sulfateand concentrated in vacuo. The crude oil was purified by flash columnchromatography (5% ether in pet.ether) to afford the required enol ether(12) as a 40:60 mixture of cis and trans isomers, (not separated) (873mg, 92%). The purity of this mixture was checked by 200 MHz nmr and themixture taken on and used in the following step without furthercharacterisation.

¹H NMR (200 MHz, CDCl₃) δ: 1.52 (2.4H,s,CCH ₃(CH ₃)), 1.62 (3.6H,s,CCH₃CH₃)), 3.49 (1.8H,s, ═OCH ₃), 3.53 (1.2H,s, OCH ₃), 3.73 (1.2H,s,NCH₃), 3.74 (1.6H,s,NCH ₃) 4.60 (0.4H,d,J=6.9 Hz,═CHOMe), 5.13(0.6H,d,J=12.7 Hz,═CHOMe), 5.78 (0.4H,d,J=6.9 Hz, CH═CHOMe), 6.32 (0.6H,d,J=12.7 Hz, CH═CHOMe), 6.83 (1H,s,═CHNH), 7.02-7.40 (3H,m,3×ArH),7.73-7.78 (1H,m,ArH).

(g) Preparation of Aldehyde (13)

To a stirred solution of enol ether (12) (854 mg, 3.73 mmol) in dioxane(40 ml) and water (10 ml) at room temperature was addedp-toluenesulfonic acid monohydrate (100 mg. 0.526 mmol), the resultingmixture was heated to 60° C. for sixteen hours. The reacton mixture wasthen diluted with water (40 ml) and extracted with ether (3×50 ml), thecombined organic extracts were washed with brine (75 ml), dried withmagnesium sulfate and concentrated in vacuo. The crude oil was purifiedby flash column chromatography (20% ether in pet.ether) to afford thedesired aldehyde (13) as an off-white solid (696.2 mg, 86%).

Mp: 39-40° C. IR (CHCl₃ soln): 1718 (s, C═O), 1615, 1546 (w, C═C). ¹HNMR (400 MHz, CDCl₃) δ: 1.55 (6H,s,CCH ₃(CH ₃)), 2.83 (2H,d,J=3.1 Hz, CH₂CHO), 3.74 (3H,s, ═NCH ₃), 6.82 (1H,s, ═CHNH), 7.10 (1H,dt,J=1.0, 7.3Hz ArH), 7.24 (1H,dt,J=1.0, 7.3 Hz, ArH), 7.32 (1H,dd,J=1.0, 7.3Hz,ArH), 7.56 (1H,dd,J=1.0, 7.3 Hz ArH), 9.51 (1H,t,J=3.1 Hz CHO). ¹³CNMR (75.3 MHz, CDCl₃) δ: 204.1, 137.9, 125.6, 125.3, 121.4, 121.3,120.7, 118.7, 109.6, 54.7, 33.6, 32.6, 29.2. LRMS (EI): 215 (M⁺, 45%),172 (100%). HRMS (EI) Calcd. for C₁₄H₁₇ON: 215.13101; found: 215.13103.Anal. Calcd for: C₁₄H₁₇ON: C, 78.10; H, 7.96; N, 6.51. Found: C, 78.22;H, 7.98; N, 6.41.

(h) Preparation of Acid (14)

To a solution of aldehyde (13) 234 mg, 1.09 mmol) in tert-butyl alcohol(6 ml) at room temperature was added 2-methyl-2-butene (8.0 ml, 2.0M inTHF, 16.3 mmol). To the resulting solution was added a solution ofsodium chlorite (148 mg, 1.63 mmol) and sodium hydrogen phoshpate (600mg, 4.36 mmol) in water (6 ml). The resulting solution was stirred fortwenty minutes at room temperature and then diluted with water (10 ml),acidified to pH 1-2 with dilute hydrochloric acid and extracted withethyl acetate (3×25 ml). The combined organic extracts were concentratedin vacuo with trace amounts of water being removed by azeotropicdistillation with toluene. The resulting crude mixture was purified byflash column chromatography (50% ether in pet.ether +1% acetic acid) toafford the acid (14) as an off white solid.

Mp: 139-140° C. IR (CHCl₃ soln): 3054, 2981 (s, br, OH), 1705 (s, C═O),1620, 1540 (w, C═C).

¹H NMR (400 MHz, CDCl₃) δ: 1.63 (6H,s,CCH ₃(CH ₃)), 2.88 (2H,c, CH₂CO₂H), 3.74 (3H,s, ═NCH ₃), 6.87 (1H,s,═CHNH), 7.10 (1H,dt,J=8.0 HzArH), 7.24 (1H,t,J=8.0 Hz, ArH), 7.32 (1H,d,J=8.0 Hz,ArH), 7.56(1H,d,J=8.0 Hz ArH). ¹³C NMR (75.3 MHz, CDCl₃) δ: 178.4, 137.7, 125.7,125.1, 122.4, 12;.2, 120.7, 118.5, 109.5, 46.8, 34.0, 32.5, 28.3. LRMS(EI): 231 (M⁺, 23%), 216 (7%), 172 (100%). HRMS (EI) Calcd. forC₁₄H₁₇O₂N: 231.12593; found: 231.12586.

(i) Preparation of Auxiliary (15)

To a stirred colled (−78° C.) solution of acid (14) (269 mg, 1.16 mol)in THF (20 ml) under argon was added triethylamine (243 μl, 1.75 mmol)and then trimethylacetyl chloride (158 μl, 1.28 mmol), the resultingmixture was warmed to 0° C., stirred for one hour and then re-coiled to−78° C. In a second flask butyllithium (1.27 ml, 1.53M in hexanes, 1.93mmol) was added drop wise to a stirred cooled (−78° C.) solution of(4S)-(−)-4-isopropyl-2-oxazolidinone (250 mg, 1.94 =mol) in THF (8 ml)under an argon atmosphere, and the resulting lithiated oxazolidinone wasadded via cannula to the reaction flask. Stirring was continued for onehour and then water (20 ml) was added and the reaction mixture wasallows to warm to room temperature whereupon it was extracted with ether(3×30 ml). The combined organic extracts were washed with brine (30 ml)and dried over magnesium sulfate, and concentrates in vacuo. The crudeyellow oil was purified by flash column chromatography (40% ether inpet.ether) to afford the desired compound (15) as an off white solid(313 mg, 78%).

IR (CHCl₃ soln): 1777, 1693 (s, C═O), 1615, 1540 (w, C═C). ¹H NMR (400MHz, CDCl₃) δ: 0.67 (3H,d,J=6.9 Hz,CH(CH ₃)CH₃), 0.77 (3H,d, J=6.9 Hz,CH(CH₃)CH ₃), 1.59 (3H,s,CCH₃(CH ₃)), 1.61 (3H,s,CCH ₃(CH₃)), 2.14(1H,m, CH(CH₃)CH₃), 3.48 (2H,s,CH ₂CON), 3.71 (3H,s, NCH ₃), 3.71(1H,br,t,J=9.0 Hz, CH _(A)H_(B)O), 3.97 (1H,dd,J=9.0,2.7 Hz CH_(A) H_(B)O), 4.18 (1H,m,CH(^(i)Pr)CH₂), 6.86 (1H,s,═CHNH), 7.07 (1H,t,J=8.0Hz ArH), 7.16 (1H,t,J=8.0 Hz, ArH), 7.24 (1H,d,J=8.0 Hz,ArH), 7.82(1H,d,J=8.0 Hz ArH). ¹³C NMR (75.3 MHz, CDCl₃) δ: 171.5, 154.0, 137.5,125.9, 125.6, 122.1, 121.0, 118.5, 109.3, 62.9, 58.5, 45.4, 35.0, 32.6,29.6, 28.7, 28.5, 17.9, 14.5. LRMS (EI): 231 (M⁺, 23%), 216 (7%), 172(100%).

(j) Preparation of Azide (16)

To a stirred, cooled (−78° C.) solution of oxazolidinone (15) (82.7 mg,0.242 mmol) in THF (3 ml) under an argon atmosphere was added potassiumbis(trimethylsilyl)amide (0.73 ml, 0.4M in THF, 0.29 mmol) and theresulting solution was stirred at −78° C. for one hour. A solution oftriisopropylsulfonyl azide (97 mg, 0.315 mmol) in THF (2 ml) pre-cooledto −78° C. was added via cannula and after one minute the reaction wasquenched by addition of glacial acetic acid (100 ml), warmed to 40° C.(water bath) and stirred for a further hour. The reaction mixture wasthen partitioned between dicloromethane (10 ml) and dilute brine (10ml), and the layers separated. The aqueous phase was extracted withdichloromethane (2×10 ml) and the combined organic extracts washed withsodium hydrogen carbonate (10 ml, sat.aq.), brine (10 ml), dried withmagnesium sulfate and concentrated in vacuo. The resulting crude oil waspurified by flash column chromatography (30% ether in pet.ether) toafford a mixture of the desired compound (16) andtrisioproylsulphonylamine (56.7 mg total, inseparable, estimated 45 mgof desired compound, approx. 50%).

¹H NMR (200 MHz, CDCl₃) δ: 0.70 (3H,d,J=6.9 Hz, CH(CH ₃)CH ₃), 0.76 (3H,d,J=6.9 Hz, CH(CH₃)CH ₃), 1.62 (3H,s,CCH₃₍CH ₃)), 1.66 (3H,s, CCH₃(CH₃)), 2.16 (1H,m, CH(CH₃)CH₃) 3.66-3.73 (6H,m,CH ₂CON, NCH ₃,CH(^(i)Pr)CH₂)), 5.66 (1H,s,CHN₃), 6.94 (1H,s,═CHNH), 7.06 (1H,t,J=8.0Hz, ArH), 7.16 (1H,t,J=8.0 Hz,ArH), 7.26 (1H,d,J=8.0 Hz,ArH), 7.75(1H,d,J=8.0 Hz, ArH).

(k) Preparation of Boc-auxiliary (17)

A mixture of azide (16) (58 mg, semi-crude, <0.152 mmol) 10% palladiumon charcoal (30 mg), and di-tert-butyl dicarbonate (66 mg, 0.304 mmol)in ethyl acetate (4 ml) was flushed with argon and then hydrogen andstirred under a hydrogen balloon for sixteen hours at room temperature.The palladium was removed via filtration through celite, the solventremoved in vacuo and the crude mixture purified by flash columnchromatography (50% ether in pet.ether) to afford the desired compound(17) (21.8 mg, <50%).

¹H NMR (200 MHz, CDCl₃) δ: 0.71 (3H,d,J=6.9 Hz,CH(CH ₃)CH ₃), 0.73(3H,d, J=6.9 Hz, CH(CH₃)CH ₃), 1.40 (9H,s,C(CH ₃)₃), 1.53 (3H,s,CCH₃(CH₃)), 1.59 (3H,s. CCH ₃(CH₃)), 2.10 (1H,m,CH(CH₃)CH₃), 2.58 (1H,m,CH_(A)H_(B)O), 3.66-3.73 (5H,m, CH _(A)H_(B)O, NCH ₃, CH(^(i)Pr)CH₂)),5.28 (1H, br, NH), 6.03 (1H, br,d,CHNHBoc), 6.98 (1H,s,═CHNH), 7.02(1H,t,J=8.0 Hz,ArH), 7.16 (1H,t,J=8.0 Hz,ArH), 7.24 (1H,d,J=8.0 Hz,ArH),7.72 (1H,d,J=8.0 Hz,ArH).

(1) Preparation of Methyl Ester of (17)

To a solution of (17) (13 mg, 0.0285 mmol) in THF (1 ml) and water (0.3ml) at 0° C. was added lithium hydroxide (8 mg, excess) as a solid inone portion. The resulting suspension was stirred at room temperaturefor sixteen hours, and then acidified with 1N hydrochloric acid, and thesolvent removed in vacuo. The resulting crude white solid was suspendedin ether and an ethereal solution of diazomethane added, until theyellow colour of the diazomethane persisted in the reaction mixture andtlc analysis showed complete consumption of starting material. Theexcess diazomethane was removed under a stream of argon and theremaining solvent removed in vacuo. The resulting crude oil was purifiedby flash column chromatography (40% ether in pet. ether) to afford thedesired compound methyl ester of (17) (7.7 mg. 75%).

¹H NMR (400 MHz, CDCl₃) δ: 1.38 (9H,s,C(CH ₃)₃), 1.47 (3H,s,CCH₃(CH ₃)),1.52 (3H,s,CCH ₃(CH₃)), 3.45 (3H,s, CO₂CH ₃), 3.72 (3 H, s, NCH ₃), 4.70(1h, d, br, NH), 5.05 (1H,d,br,CHNHBoc), 6.81 (1H,s,═CHNH), 7.07(1H,t,J=8.0 Hz ArH), 7.21 (1H,t,J=8.0 Hz, ArH), 7.30 (1H,d,J=8.0Hz,ArH), 7.80 (1H,d,J=8.0 Hz ArH).

(m) Alternative Route to Methyl Ester of (17)

Reversal of the above two steps allows for the synthesis of the samecompound in comparable yield.

(n) Preparation of Methylated Methyl Ester

To a suspension of sodium hydride (excess) and methyl iodide (excess) indry THF (0.5 ml) under an argon atmosphere was added the methyl ester of(17) (10.4 mg 0.0289 mmol) in THF (1 ml) via cannula and the resultingmixture was stirred for sixteen hours at room temperature. Water wasadded and the mixture was extracted with ether, the combined organicextracts were washed with brine, dried with magnesium sulfate andconcentrated in vacuo. The crude oil was purified by flash columnchromatography (40% ether in pet.ether) to afford the desiredN-methylated methyl ester as a colourless oil (3.1 mg, approx. 30%).

¹H NMR (400 MHz, CDCl₃) δ: 1.42 (9H,s,C(CH ₃)₃), 1.52 (3H,s,CCH₃(CH₃)),1.64 (3H,s,CCH ₃(CH₃)), 2.70 (3H,s, (split) NCH ₃Boc), 3.45 (3H,s,split, CO₂CH ₃), 3.71 (3H,s,NCH ₃), 5.40 (1H,s,split, CHNCH₃Boc), 6.98(1H,s,═CHNH), 7.00-7.25 (3H,m, ArH), 7.72 (1H,d,br,split,ArH).

(o) Preparation of (18)

The procedure of step (1) was followed, but the esterification withdiazomethane was omitted.

(p) Preparation of (19)

N-Boc-amine acid (18) (10 mmol) and methyl iodide (5 ml; 80 mmol) weredissolved in THF (30 ml) and the solution was cooled to 0° C. underargon. Sodium hydride dispersion (1.32 g; 30 mmol) was added cautiouslywith gentle stirring. The suspension was stirred at room temperature for16 h. Ethyl acetate (50 ml) was then added (to consume the sodiumhydroxide formed from the excess sodium hydride), followed by water,drop wise, to destroy the excess sodium hydride. The solution wasevaporated to dryness, and the oily residue partitioned between ether(30 ml) and water (100 ml). The ether layer was washed with 5% aqueousNaHCO₃ (50 ml) and the combined aqueous extracts and acidified to pH 3with aqueous citric acid. The product was extracted into ethyl acetate(3×25 ml), the extract washed with water (2×25 ml), 5% aqueous sodiumthiosulphate (2×25 ml; to remove iodine), and water (2×25 ml), driedover MgSO₄ and evaporated to give a pale yellow oil of (19).

3. Coupling of Amino Acids

N-Boc amino acid (19) (1 mmol), amino acid methyl ester of moiety B (1.1mmol) and coupling agent py-BOP (1.1 mmol) were dissolved in CH₂Cl₂ (10ml) under argon. TIEA (3 mmol) was added and the reaction was stirredfor 1 h at room temperature. Excess solvents were removed in vacuoyielding a yellow oily residue which was redissolved in EtOAc (50 ml).Washing the EtOAc solution with 10% citric acid (2×25 ml), water (25ml), 5% NaHCO₃ (2×25 ml), water (25 ml), following -by drying overanhydrous MgSO₄ and normal phase silica gel chromatography afforded theprotected peptide A-B as a white amorphous solid.

Coupling of the peptide A-B to amino acid moiety of C (5) was carriedout in a similar way.

Testing of Compounds

-   1. The cytotoxicity of compounds described herein have been tested    as described in J. Immunol. Methods, 65, 55-63, 1983 and the results    are provided in table 4 below, wherein P388, refers to in vitro test    versus murine leukaemia P388, U373, refers to in vitro human    glioblastoma/astrocytoma U373, HEY refers to in vitro human ovarian    carcinoma HEY, MCF7 refers to in vitro human breast cancer MCF7.-   2. In in vivo test as described in NIH Publications No. 84-2635, “In    Vivo Cancer Models”, Developmental Therapeutic Program, Division of    Cancer Treatment, National Cancer Institute, Bethesda, Md.    hemiasterlin has been found to be cytotoxic. In mice injected with    1×10⁶ P388 leukaemia cells, hemiasterlin resulted in a %TC of 223    after 5 daily doses of 0.45 μg begun 24 hours after implantation.    There were several long term survivors in the experiment.

TABLE 4 IC₅₀ Values (μg/ml) cell compound P388 U373 HEY MCF7 mitosis

4.57 ×10⁻⁵ 1.2 ×10⁻² 1.4 ×10⁻³ 1.58 ×10⁻⁴ 1.58 ×10⁻⁴

1.71 ×10⁻⁶ 1.5 ×10⁻³ 7.6 ×10⁻³ 1.54 ×10⁻³ 1.02 ×10⁻³

 7.0 ×10⁻³ 1.6 ×10⁻² 1.50 ×10⁻² 9.96 ×10⁻³

 7.3 ×10⁻³ 0.27 0.19 6.8 Geodiamolide G (compound I) 7.7  8.6 

 5.4 ×10⁻³ 2.16 ×10⁻⁴

1.06 ×10⁻³

0.1

-   3. Compounds described herein were comparatively tested for their    antimitotic activity against human mammary carcinoma MCF7 cells.

MCF7 cells were grown as a monolayer in RPMI supplemented with 15% fetalcalf serum and antibiotics at 37° C. in humidified 10% CO₂. Allcompounds were dissolved in dimethyl sulfoxide except for vinblastine (aknown drug) which was a 1 mg/ml solution in physiological saline.Exponentially growing MCF7 cells were treated with different drugconcentrations for 20 h, prepared for chromosome spreads, and thepercentage of mitotic cells determined by fluorescence microscopy. Theresults are shown in FIGS. 1 and 2. Hemiasterlin, Hemiasterlin A andmodified compounds were very potent antimitotic agents, with IC₅₀ valuesof 0.3 nM and 3 nM respectively. Hemiasterlin and Hemiasterlin A weremore potent than Taxol, Vinblastine and Nocodazole (all known drugs).

The effect of Hemiasterlin and Hemiasterlin A on the morphology of theirmitotic spindles was examined by indirect immunofluorescence using amonoclonal antibody to β-tubulin and the distribution of theirchromosomes using the fluorescent DNA dye bisbenzimide. In the presenceof hemiasterlin A at 2 nM no completely normal spindles were seen. Somecells showed relatively minor abnormalities in which a bipolar spindlewas present but the astral microtubules were considerably longer thannormal and the chromosomes were not completely confined to the metaphaseplate. Most commonly cells had multiple asters, and the chromosomes weredistributed in a spherical mass. Half-maximal concentrations of taxol,vinblastine and nocodazole produced the same types of abnormal spindleas hemiasterlin A. Hemiasterlin A at 10 nM, the lowest concentrationcausing maximal mitotic arrest in MCF7 cells, caused microtubuledepolymerisation in mitotic cells. This was also the case for highconcentrations of vinblastine and nocodazole. Taxol at highconcentrations had a quite different effect, causing bundling ofcytoplasmic microtubules in interphase cells and very dense multipleasters in mitotic cells.

These results show that Hemiasterlins cause mitotic arrest and produceabnormal mitotic spindles. They can be used in lieu of other antimitoticdrugs in procedures that require blocking cells in mitosis, such as thepreparation of mitotic spreads for karyotype analysis. They can also beused to probe microtubule function in mitotic cells.

1. A compound of general formula I

wherein: R₁, R₂, and R₇₀ independently represent a hydrogen atom or anoptionally substituted alkyl or acyl group, wherein the alkyl or acylgroup has up to 8 carbon atoms, and wherein the optional substituentsare selected from the group consisting of halogen atoms and nitro,cyano, alkoxy, hydroxy, amino, alkylamino, sulfinyl, alkylsulfinyl,sulfonyl, alkylsulfonyl, amido, alkylamido, alkylcarbonyl,haloalkoxycarbonyl, and haloalkyl groups; Y represents an optionalsubstituent; n represents 0, 1, 2, 3, or 4; R₃ represents a hydrogenatom, or an optionally substituted alkyl group; R₇₄ represents ahydrogen atom, a hydroxy group or an optionally substituted alkyl oracyl group; R₇ represents a hydrogen atom or an alkyl group; R₇₅represents an optionally substituted alkyl group up to 6 carbon atoms;and i) R₇₁ represents a hydrogen atom or an optionally substituted alkylor acyl group; and R₇₂ represents a hydrogen atom; or ii) R₇₁ and R₇₂each represent radicals so that a double bond is formed between thecarbon atoms to which they are attached.
 2. The compound of claim 1,wherein n represents
 0. 3. The compound of claim 1, wherein R₇₁represents a hydrogen atom or an alkyl or acyl group.
 4. A compound ofgeneral formula I

wherein: R₁, R₂, and R₇₀ independently represent a hydrogen atom or anoptionally substituted alkyl or acyl group, wherein the alkyl or acylgroup has up to 8 carbon atoms, and wherein the optional substituentsare selected from the group consisting of halogen atoms and nitro,cyano, alkoxy, hydroxy, amino, alkylamino, sulfinyl, alkylsulfinyl,sulfonyl, alkylsulfonyl, amido, alkylamido, alkylcarbonyl,haloalkoxycarbonyl, and haloalkyl groups; Y represents an optionalsubstituent; n represents 0, 1, 2, 3, or 4; R₃ represents a hydrogenatom, or an optionally substituted alkyl group; R₇₄ represents ahydrogen atom, a hydroxy group or an optionally substituted alkyl oracyl group; R₇ represents a hydrogen atom or an alkyl group; R₇₅represents a group of general formula II:

wherein Q represents an optionally substituted —CH₂—, CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CHCH—, —CH₂C≡C—, or phenylene moiety; and X representsa group —OR₈, —SR₈, or —NR₉R₁₀, wherein R₈ independently represents ahydrogen atom or an optionally substituted alkyl group, and R₉ and R₁₀independently represent a hydrogen atom or an optionally substitutedalkyl group having up to 8 carbon atoms; and i) R₇₁ represents ahydrogen atom or an optionally substituted alkyl or acyl group; and R₇₂represents a hydrogen atom; or ii) R₇₁ and R₇₂ each represent radicalsso that a double bond is formed between the carbon atoms to which theyare attached.
 5. The compound of claim 4, wherein X represents a group—OR₈, wherein R₈ represents a hydrogen atom or a methyl group.
 6. Thecompound of claim 4, wherein X represents a group —NR₉R₁₀, wherein R₉represents a hydrogen atom or an alkyl group having up to 8 carbonatoms, and R₁₀ represents a substituted alkyl group having up to 8carbon atoms.
 7. The compound of claim 1, wherein the compound hasantimitotic activity.
 8. A compound of general formula I

wherein R₁ represents a hydrogen atom; R₂ represents a hydrogen atom, oran alkyl group, or an acyl group; R₃ represents a hydrogen atom, or anoptionally substituted alkyl group; Y represents an optionalsubstituent; n represents 0; R₇₀ and R₇₁ independently represent ahydrogen atom or an optionally substituted alkyl group; R₇₂ and R₇₄represent hydrogen atoms; R₇₄ represents an optionally substituted alkylor acyl group; R₇ represents a hydrogen atom or an alkyl group; R₇₅represents a group of general formula III,

wherein R₄ represents a hydrogen atom, or an optionally substitutedalkyl group; R₅ represents a hydrogen atom or an alkyl group; R₇₆ andR₇₇ each represent a hydrogen atom or a radical so that a double bond isformed between the carbon atoms to which they are attached; and Xrepresents a group —OR₈ or a group —NR₉R₁₀, wherein R₈, R₉ and R₁₀independently represent a hydrogen atom or an optionally substitutedalkyl group.
 9. The compound of claim 8, wherein R₇₀ and R₇₁ eachrepresent a methyl group.
 10. A compound of general formula I

wherein R₁ represents a hydrogen atom or an alkyl group; R₂ representsan acyl group; R₃ represents a hydrogen atom, or an optionallysubstituted alkyl group; Y represents an optional substituent; nrepresents 0; R₇₀ and R₇₁ independently represent a hydrogen atom or anoptionally substituted alkyl group; R₇₂ and R₇₄ represent hydrogenatoms; R₇₄ represents an optionally substituted alkyl or acyl group; R₇represents a hydrogen atom or an alkyl group; R₇₅ represents a group ofgeneral formula III,

wherein R₄ represents a hydrogen atom, or an optionally substitutedalkyl group; R₅ represents a hydrogen atom or an alkyl group; R₇₆ andR₇₇ each represent a hydrogen atom or a radical so that a double bond isformed between the carbon atoms to which they are attached; and Xrepresents a group —OR₈ or a group —NR₉R₁₀, wherein R₈, R₉ and R₁₀independently represent a hydrogen atom or an optionally substitutedalkyl group.
 11. The compound of claim 10, wherein R₇₀ and R₇₁ eachrepresent a methyl group.